1. Field of the Invention
The present invention relates to a novel method for measurement of the true activity of tissue plasminogen activator (hereafter referred to as t-PA) in blood and a device and kit as well as a novel monoclonal antibody used for the measurement. More particularly, the present invention relates to a method for measurement of the true activity of t-PA in blood which comprises using a solid phase having bound thereto anti t-PA-antibody IgG fraction having affinity to a site other than active site of t-PA and having specificity to t-PA in whole blood or diluted blood from which plasma is not separated as a sample, and measuring activator activity in the product of the solid phase and t-PA in the sample.
2. Description of the Prior Art
t-PA is one of serine protease which takes part in fibrinolytic mechanism. It is known that its action is to prevent formation of thrombus by dissolving and removing fibrin formed in blood vessel or tissue. Further, t-PA is synthesized in blood endothelial cells and, secreted and released depending upon necessity and is thus a factor that is essential to prevention of thrombus formation and maintaining blood flow. From such, it is easily presumed that a high value of t-PA activity in blood would have a tendency to bleed and a low value would have a tendency to clot blood. However, so far no appropriate method has been developed yet, due to the presence of inhibitors(PAI) to t-PA in blood and to short half life of t-PA. In addition to those, t-PA can be readily inactivated under ice cooling even in plasma separated from blood. According to the results obtained in conventional methods for measurement, however, examples of clinical tests showing high t-PA levels are reported to be hepatic disorder [J. Clin. Invest., 43, 681 (1964), J. Clin. Pathol., 37, 772 (1983)], ovarian cancer [Eur. J. Cancer Clin. Oncol., 20, 577 (1984)], cerebral hemorrhage [Blood, 61, 267 (1983)]; regarding patients with low t-PA levels, there are reports on deep venous thrombus [Br. J. Surg., 70, 369 (1983), British Med. J., 290, 1453 (1985)], Atrophic Blanche [Arch. Pathol. Lab. Med., 110, 517 (1986)], endometrial malignant tumor [Cancer, 48, 1484 (1982)], myocardial infarction or hyperlipidemia [The New England Journal of Medicine, 313, No. 25, 1557 (1985)], etc. In addition, there is a report on the family line in which venous thrombus is repeated due to abnormality in t-PA production mechanism [Acta. Chim. Scand., 138, 313 (1972), Acta. Med. Scand., 203, 477 (1978)].
From the foregoing, it is also very important from the clinical aspect to know t-PA levels in blood.
In general, t-PA level in plasma has been hitherto determined by its amount of antigen and its activity. For determination of amount of antigen, RIA (radioimmunoassay), IRMA(immuno radiometric assay), ELISA (Enzyme linked-immunosorbent assay), etc. using anti t-PA antibody have been used (Published Unexamined Japanese Patent Application Laid Open Nos. 59-174759 and 61-148200).
On the other hand, the measurement of activity is roughly classified into two methods; namely, fibrinolysis and synthetic substrate method. For the fibrinolysis method, the following publications are given.
Thromb. Haemostas., 52 (1), 19 (1984) PA0 Thromb. Haemostas., 45 (2), 107 (1981) PA0 Thromb. Diathes. Haemoth., 32, 356 (1974) PA0 Thrombosis Res., 43, 129 (1986) PA0 Thrombosis Haemostasis, 53 (3), 356 (1985) PA0 Thrombosis Res., 46, 213 (1987) PA0 Clinica. Chimica. Acta., 127, 279 (1983)
For the synthetic substrate method, the following publications are given.
Further as materials for examining t-PA activity in blood, plasma is usually employed and for purposes of removing inhibitors to t-PA, various treatments are conducted. For example, there is a method which comprises centrifuging blood for 5 minutes, then separating plasma from the blood, immediately adjusting pH to 5.9 with acetic acid to precipitate Euglobulin fraction and centrifuging the precipitates [Thromb. Haemostas., 48 (3), 266 (1982)], a method which comprises adjusting pH of plasma to 3.9 and instantaneously freezing the plasma at -70.degree. C. (dry ice) (which is dissolved upon use with heating and its pH is readjusted to about 7.4) and a method which comprises adsorbing plasma t-PA to Lysine Sepharose and washing to remove t-PA inhibitor [Thrombosis Res., 46, 413 (1987)].
In each of the methods for determining the antigen level of t-PA, however, the reaction product of t-PA with its inhibitor is also measured as the amount of antigen but each method fails to measure true t-PA activity.
In addition, the conventional methods described above further involve serious problems in that:
(1) a time period necessary for centrifugation in order to separate plasma from blood takes at least 10 minutes from the start to the end of the operation, during which t-PA activity in plasma is greatly lost; PA1 (2) it takes a long time until the result is judged (for about 20 hours ), which not only lacks simplicity but also fails to measure true t-PA activity, because t-PA inhibitor is continuously affecting the result during the reaction time, depending upon method; etc.
Furthermore, conventional methods require dilution from several ten to several thousand-fold thereby to avoid effects of t-PA inhibitor and thus encounter a problem that even t-PA activity cannot be measured, unless the methods are excellent in their sensitivity and accuracy.
From the foregoing reasons, all of the methods that can determine t-PA activity in blood as it is have not been successful.
Therefore, in order to establish a method for measurement of true t-PA activity, the present inventors have made extensive investigations on such a method for measuring t-PA activity in whole blood or diluted blood without centrifugation that inactivates the t-PA activity. As a result, they have found that true t-PA activity in a sample can be determined at high accuracy and sensitivity in an extremely simple treatment for a short time by the methods of either acting t-PA specific chromogenic peptide substrate directly on the reaction product obtained by reacting anti t-PA monoclonal antibody coupled with solid phase, or acting enzyme substrate specific to t-PA followed by reacting specific substrate to the plasmin produced by this reaction and, have come to reach the present invention.